Keller H, Altenhein B, Gebauer D, Richter S, Stricker S, Markl J
Institute of Zoology, Johannes Gutenberg University of Mainz, Germany.
Eur J Biochem. 1999 Aug;264(1):27-38. doi: 10.1046/j.1432-1327.1999.00564.x.
We have identified two separate hemocyanin types (HtH1 and HtH2) in the European abalone Haliotis tuberculata. HtH1/HtH2 hybrid molecules were not found. By selective dissociation of HtH2 we isolated HtH1 which, as revealed by electron microscopy and SDS/PAGE, is present as didecamers of a approximately 400 kDa subunit. Immunologically, HtH1 and HtH2 correspond to keyhole limpet hemocyanin (KLH)1 and KLH2, respectively, the two well-studied hemocyanin types of the closely related marine gastropod Megathura crenulata. On the basis of limited proteolytic cleavage, two-dimensional immunoelectrophoresis, SDS/PAGE and N-terminal sequencing, we identified eight different 40-60 kDa functional units in HtH1, termed HtH1-a to HtH1-h, and determined their linear arrangement within the elongated subunit. From Haliotis mantle tissue, rich in hemocyanin-producing pore cells, we isolated mRNA and constructed a cDNA library. By expression screening with HtH-specific rabbit antibodies, a cDNA clone was isolated and sequenced which codes for the three C-terminal functional units f, g and h of HtH1. Their sequences were aligned to those available from other molluscs, notably to functional unit f and functional unit g from the cephalopod Octopus dofleini. HtH1-f, which is the first sequenced functional unit of type f from a gastropod hemocyanin, corresponds to functional unit f from Octopus. Also functional unit g from Haliotis and Octopus correspond to each other. HtH1-h is a gastropod hemocyanin functional unit type which is absent in cephalopods and has not been sequenced previously. It exhibits a unique tail extension of approximately 95 amino acids, which is lacking in functional units a to g and aligns with a published peptide sequence of 48 amino acids from functional unit h of Helix pomatia hemocyanin. The new Haliotis sequences are discussed with respect to their counterparts in Octopus, the 15 A three-dimensional reconstruction of the KLH1 didecamer from electron micrographs, and the recent 2.3 A X-ray structure of functional unit g from Octopus hemocyanin.
我们在欧洲鲍鱼皱纹盘鲍中鉴定出两种不同的血蓝蛋白类型（HtH1和HtH2）。未发现HtH1/HtH2杂交分子。通过对HtH2进行选择性解离，我们分离出了HtH1，电子显微镜和SDS/PAGE显示，HtH1以约400 kDa亚基的双十聚体形式存在。在免疫学上，HtH1和HtH2分别对应于匙孔血蓝蛋白（KLH）1和KLH2，这是密切相关的海洋腹足纲动物巨紫螺中两种经过充分研究的血蓝蛋白类型。基于有限的蛋白水解切割、二维免疫电泳、SDS/PAGE和N端测序，我们在HtH1中鉴定出八个不同的40 - 60 kDa功能单元，命名为HtH1-a至HtH1-h，并确定了它们在细长亚基中的线性排列。从富含产生血蓝蛋白的孔细胞的鲍鱼外套膜组织中，我们分离出mRNA并构建了一个cDNA文库。通过用HtH特异性兔抗体进行表达筛选，分离并测序了一个cDNA克隆，该克隆编码HtH1的三个C端功能单元f、g和h。将它们的序列与其他软体动物的序列进行比对，特别是与头足纲动物杜氏枪乌贼的功能单元f和功能单元g进行比对。HtH1-f是来自腹足纲动物血蓝蛋白的f型第一个测序的功能单元，与枪乌贼的功能单元f相对应。鲍鱼和枪乌贼的功能单元g也相互对应。HtH1-h是一种腹足纲动物血蓝蛋白功能单元类型，在头足纲动物中不存在，且此前未进行过测序。它具有约95个氨基酸的独特尾部延伸，这在功能单元a至g中不存在，并且与已发表的来自苹果螺血蓝蛋白功能单元h的48个氨基酸的肽序列一致。结合枪乌贼中对应序列、电子显微镜下KLH1双十聚体的三维重建以及最近枪乌贼血蓝蛋白功能单元g的2.3 Å X射线结构，对新的鲍鱼序列进行了讨论。
